Abstract

Synaptic transmission from Drosophila photoreceptors to lamina neurons requires recycling of histamine neurotransmitter. Synaptic histamine is cleared by uptake into glia and conversion into carcinine, which functions as transport metabolite. How carcinine is transported from glia to photoreceptor neurons remains unclear. In a targeted RNAi screen for genes involved in this pathway, we identified carT, which encodes a member of the SLC22A transporter family. CarT expression in photoreceptors is necessary and sufficient for fly vision and behavior. Carcinine accumulates in the lamina of carT flies. Wild-type levels are restored by photoreceptor-specific expression of CarT, and endogenous tagging suggests CarT localizes to synaptic endings. Heterologous expression of CarT in S2 cells is sufficient for carcinine uptake, demonstrating the ability of CarT to utilize carcinine as a transport substrate. Together, our results demonstrate that CarT transports the histamine metabolite carcinine into photoreceptor neurons, thus contributing an essential step to the histamine–carcinine cycle.

eLife digest

Photoreceptors are light-sensitive neurons in the eyes of the fruit fly Drosophila that form connections with other neurons in the fly’s brain. At these connections, which are called synapses, the photoreceptors continuously release a chemical called histamine.

Photoreceptors will release more or less histamine depending on changes in light intensity, but always tend to release more histamine than they can produce themselves from scratch. This means that the visual system in Drosophila relies on a pathway that recycles histamine. That is to say, glial cells (which support the activity of the neurons) remove the chemical from synapses and return it to the photoreceptor neurons in a slightly modified form called “carcinine”. The photoreceptors then quickly convert the chemical back into histamine, ready to be released.

Stenesen et al. set out to identify the proteins that support this recycling pathway, and started by screening around 130 genes that encode transporter proteins for potential roles in histamine recycling. This screen identified a gene encoding a protein that was named CarT. This protein transports carcinine, the modified version of the histamine neurotransmitter.

Stenesen et al. show that the photoreceptor neurons make the CarT protein and need this protein to take up the carcinine released by the supporting glial cells. Without CarT, photoreceptor neurons cannot transmit visual information, and so mutant flies in which the gene for CarT is deleted are blind. Follow-up studies related to this work could involve identifying the transporters that move histamine and carcinine in and out of the glia cells, and exploring what other neurons and behaviors in fruit flies rely on CarT’s activity.

Results and discussion

The Drosophila genome is estimated to contain 603 transmembrane transporters (Ren et al., 2007; Featherstone, 2011). To concentrate on those transporters with a higher probability of participating in histamine neurotransmitter recycling, we focused on families that contain individual members previously associated with neurotransmitter transport. This includes known serotonin, dopamine, gamma-aminobutyric acid, and glutamate neurotransmitter transporters in the solute-linked carrier (SLC) families SLC1 and SLC6, and other transporters in the SLC17, SLC18 and SLC22A families (César-Razquin et al., 2015). In addition, ATP-binding cassette transporters have been implicated in altered neurotransmitter distribution (Borycz et al., 2008). In Drosophila, these transporter families contain 137 members that we considered as candidates potentially involved in the histamine–carcinine cycle. To test their possible roles in photoreceptor neurons, we knocked down each candidate gene individually using Glass Multiple Response element (GMR)-Gal4 to drive double-stranded RNA expression in photoreceptors and evaluated visual signal transduction by ERG recordings.

The screen yielded a single transporter gene, CG9317, whose knockdown caused severely reduced ON and OFF transients (Figure 1A). To test the consistency of this result in multiple genetic backgrounds and confirm its specific requirement in photoreceptor neurons, we expressed the CG9317 RNAi transgene with two additional photoreceptor-specific Gal4 lines, longGMR-Gal4 (Wernet et al., 2003) and otd1.6-Gal4 (McDonald et al., 2010) and the pan-glial driver, repo-Gal4 (Sepp et al., 2001). ERG recordings indicated that CG9317 is necessary in photoreceptor neurons, but not glia, for proper histaminergic transduction in the visual system (Figure 1A–C). Of note, compared with wild type, none of these genotypes displayed significantly reduced sustained negative potentials ( Figure 1D), indicating that CG9317 knockdown did not affect overall photoreceptor health (Williamson et al., 2010). Based on these results and the findings presented below, we will refer to CG9317 as carcinine transporter, abbreviated carT.

Photoreceptor-specific expression of CarT is sufficient for visual signal transduction

To test whether carT is the causal gene, the knockdown of which is responsible for loss of ERG ON and OFF transient components, we generated null alleles by clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated mutagenesis (Gratz et al., 2013; Bassett et al., 2013; Yu et al., 2013). We isolated three individual CRISPR-induced deletions: 11 bp for carT16A, 26 bp for carT16B and 56 bp for carT43 (Figure 2A). Each of these mutations resulted in a frameshift within the first transmembrane domain (Figure 2B) followed by a premature stop codon within 17, 19 and 1 bp, respectively. All three carT alleles were homozygous viable and fertile, but lacked the ON and OFF transients, indicating that carT is required for visual signal transduction (Figure 2C,D). For further analysis, we focused on the allele with the largest deletion, carT43. To prove that loss of CarT function causes the ERG-phenotype in carT43 mutants, we tested whether it could be rescued by cell type-specific expression of the CarT transcript. When driven in carT43 mutant photoreceptor neurons by longGMR-Gal4, CarT restored ON and OFF transient components of ERG recordings (Figure 2C,D). By contrast, glial-specific expression of CarT failed to rescue ON and OFF transient defects (Figure 2C,D). To further probe CarT function, we generated a Myc-CarT transgene. This N-terminally tagged transporter was fully functional, as its expression in carT43 also rescued defects in ON and OFF transients (Figure 2C,D). Taken together, these data indicate that expression of CarT in photoreceptor neurons is necessary and sufficient for visual signal transduction.

CarT is required in photoreceptors but not glia for normal cellular and behavioral visual responses.

(A) CRISPR targeted site in the carT gene and resulting mutations. (B) Primary structure and predicted transmembrane domains (yellow) of CarT. Red arrows indicate location of premature stops within mutant alleles. Gray and black letters indicate conserved and highly conserved amino acids among SLC22 transporters (Eraly et al., 2004; Koepsell, 2013), respectively. The SLC22 family is characterized by a large extracellular loop containing four cysteines (C) and a glycosylation site (blue) at conserved positions. (C) ERGs recorded from female flies carrying wild type (+) or the indicated CarT alleles (16A, 16B, 43, or tdTomato-CarT) with or without expression of a wild-type or a Myc-tagged UAS-CarT transgene driven by longGMR-Gal4 or repo-Gal4 as indicated. (D) Quantifications of ON and OFF transients, and SNPs of all genotypes in panel C averaged from three replicate experiments, including at least 45 traces from 15 flies (G: longGMR-Gal4; R: repo-Gal4; -: not present). (E) Phototactic behavior of OreR and carT43 mutant flies compared with other mutants that disrupt the histamine–carcinine cycle (HdcMB07212, ebony1, or tan1) presented as a light preference index. (F) Phototactic behavior of flies expressing a UAS-CarT transgene in photoreceptor neurons under control of the longGMR-Gal4 driver in carT43 mutants compared with controls shows the restoration of wild type behavior. For each graph, the box outlines the upper and lower quartiles, and the whiskers show minimum and maximum recorded values. ns, not significant; ****p < 0.0001 compared with carT43 (D) or OreR (E and F). CRISPR, clustered regularly-interspaced short palindromic repeats; ERG, electroretinograms; SNPs, sustained negative potentials.

Photoreceptor CarT is required for normal visual behavior

To examine whether the disruption of visual neurotransmission in carT mutants alters visual behavior, we used a phototaxis assay (Benzer, 1967). Wild-type flies were naturally phototactic (Figure 2E). However, flies with defects in the synthesis of histamine (HdcMB07212) or its recycling (ebony1 or tan1) were randomly distributed between the lit and dark arms of a T-maze, consistent with a lack of phototactic preference due to their inability to detect light (Figure 2E). Similarly, phototactic behavior was severely reduced in carT43 flies (Figure 2E). Light preference of carT43 flies was restored by expression of wild-type CarT in photoreceptor neurons under control of longGMR-Gal4 (Figure 2F). Together, these results indicate that CarT expression in photoreceptor neurons is necessary and sufficient for behavioral aspects of normal vision and electrophysiological properties of visual transduction.

Carcinine accumulates in CarT mutant lamina

Interference with aspects of the histamine–carcinine cycle by different mutations causes distinct changes in the distribution of these two metabolites in the retina and lamina, although overall architecture of these tissues is not altered in carT, ebony, tan and Hdc mutants (Figure 3A–D) and references (Chaturvedi et al., 2014; Borycz et al., 2008). Consistent with their function in the histamine–carcinine cycle, characteristic increases in the levels of histamine in ebony1 (Figure 3C) and carcinine in tan1 heads (Figure 3D) were detected by antibody staining (Chaturvedi et al., 2014). When compared with wild type, carT43 heads displayed no discernable alterations in histamine staining intensity or distribution (Figure 3C). However, carcinine levels were elevated in carT43 lamina (Figure 3D, arrow) and medulla (arrowhead). Staining for carcinine in carT43 lamina partially localized to the glia visualized by repo-Gal4 driven UAS-mCD8::RFP (Figure 3E) suggested a build up of carcinine in the glia and the extracellular space in the lamina, consistent with a requirement of CarT in carcinine transport into photoreceptors. Notably, carcinine accumulation in carT43 brains was restored to wild-type levels by photoreceptor-specific expression of a Myc-CarT transgene (Figure 3D).

Loss of CarT in photoreceptors increases laminal carcinine.

(A) Confocal sections of photoreceptor axonal endings within the lamina stained for the synapse marker Bruchpilot (Brp) and the glia-specific Ebony. (B) Micrographs of cryo-sections from control (OreR) and flies expressing the UAS-Myc-CarT transgene driven by longGMR-Gal4 in the carT43 background. Retina (R), lamina (L), and medulla (M) neuropiles are stained for DNA (magenta) and Myc-tagged CarT (green). Arrow points to Myc staining in photoreceptor axonal endings and arrowhead to distal retina. (C,D) Micrographs of cryo-sections from control (OreR) and mutant flies affecting the histamine–carcinine cycle: carT43, carT43;GMR-Gal4/UAS-Myc-CarT, ebony1, tan1, and HdcMB07212 stained for histamine (C) or carcinine (D). Arrow and arrowhead in D point to carcinine accumulations in the lamina and medulla, respectively. (E) Micrographs from control (carT43/+ ) or carT43 flies expressing a UAS-mCD8::RFP transgene driven by repo-Gal4 and stained for carcinine. (F) Micrographs showing tdTomato fluorescence of the in-frame tdTomato-CarT allele compared with wild type control. DNA staining is shown in green. Arrowheads point to tdTomato signal at R7 and R8 photoreceptor terminals within the medulla. (G) Confocal section of laminal region of a tdTomato-CarT fly stained for glia-specific Ebony. (H) Confocal sections of flies heterozygous for tdTomato-CarT (red) and photoreceptor-specific 3xPax3-eGFP (blue) stained for Brp (green). Sections are parallel to and across photoreceptor axons, respectively. Scale bars are 20 µm in A, E, G and H and 50 µm in B–D and F.

CarT is enriched close to photoreceptor synapses

Within photoreceptors, the longGMR-driven Myc-CarT transporter was enriched in their axonal endings in the lamina (arrow in Figure 3B) and the distal retina (arrowhead in Figure 3B) indicating CarT may function near synaptic terminals and in neuronal cell bodies to facilitate perisynaptic and long-distance recycling, two pathways previously suggested (Chaturvedi et al., 2014; Borycz et al., 2012; Rahman et al., 2012). Endogenous CarT RNA expression is highly enriched in the eye (http://flybase.org/reports/FBlc0000157.html). To further examine endogenous CarT expression, we utilized CRISPR-induced deletions coupled with homology-directed repair to incorporate an in-frame insertion of tdTomato N-terminally to the CarT coding sequence (Figure 2B). ERG analysis of flies harboring one copy of this tdTomato-CarT over the nonfunctional carT43 allele displayed normal ON and OFF transient responses, indicating that the endogenous tdTomato-tagged CarT was fully functional (Figure 2C,D). This endogenous tdTomato-CarT was highly enriched within photoreceptor projections in the lamina and medulla (Figure 3F). Co-labeling with the glial Ebony (Figure 3G), or synaptic Bruchpilot and a 3xPax3-eGFP, which labels photoreceptor neurons (Figure 3H), indicates that CarT is present in photoreceptors near synaptic terminals (Figure 3H).

CarT transports carcinine

To determine whether the CarT transporter utilizes carcinine as a substrate, we developed an immunofluorescence-based carcinine transport assay. Full-length or internally truncated Myc-CarT were expressed in S2 cells that were probed by antibody staining for carcinine uptake. When cultured in standard Schneider’s medium, transfected S2 cells displayed no appreciable carcinine staining, indicating a lack of endogenous carcinine and sufficient specificity of the carcinine antibody in this context (Figure 4A and A’; quantified in D). When culture media was supplemented with carcinine (0.5 mM), cells expressing full-length Myc-CarT stained positively for carcinine, indicating its uptake in these cells. Importantly, neighboring untransfected cells (negative for the Myc epitope) lacked carcinine staining, despite being maintained in the same carcinine-supplemented medium as Myc-CarT positive cells (Figure 4B and B’). Furthermore, an internal deletion of 10 of the 12 transmembrane domains within CarT abolished carcinine uptake (Figure 4C and C’; quantified in D). Taken together, these data demonstrate CarT functions as a carcinine transporter.

Sequence comparison indicates that CarT is a member of the SLC22 family of transporters (Eraly et al., 2004) and together with other CarT-like transporters in invertebrates constitutes a distinct subfamily (Figure 2—figure supplement 1). Interestingly, OCT2 and OCT3, members of the closely related organic cation subfamily of SLC22A transporters, have been implicated in histamine uptake into human astrocytes (Yoshikawa et al., 2013) and the transport of histamine receptor antagonists (Koepsell, 2013). Many of the biochemically characterized members of the extended SLC22 family that are involved in the transport of neurotransmitters, exhibit overlapping substrate specificity providing transport redundancies (Koepsell, 2013). However, our data argue against such redundancy as carT null alleles efficiently blocks synaptic transmission dependent on the histamine–carcinine cycle (Figure 4—figure supplement 1). A role in this process has previously been suggested for the putative Na/Cl– dependent neurotransmitter/osmolyte transporter Inebriated (Stuart et al., 2007), possibly by directly transporting carcinine into photoreceptors (Gavin et al., 2007). No direct evidence for such a function has been reported, however, suggesting that Inebriated may indirectly support the histamine–carcinine cycle by promoting the long-distance recycling of β-alanine from photoreceptors to glia (Stuart et al., 2007; Chaturvedi et al., 2014; Borycz et al., 2012). An indirect role of Inebriated is also more consistent with its role in water homeostasis in the hindgut (Luan et al., 2015).

Here, we presented several lines of evidence that CG9317, which is highly expressed in heads, but not bodies (Eraly et al., 2004), encodes the carcinine transporter CarT. Heterologous expression of CarT in cultured cells was sufficient to facilitate carcinine uptake. This biochemical activity and the accumulation of carcinine in CarT lamina could be consistent with CarT facilitating export of carcinine from glia, or its import into photoreceptors. Strong support for the second possibility is provided by genetic experiments that reveal a requirement of CarT in photoreceptors, but not glia and the accumulation of carcinine in the lamina. CarT expression in photoreceptors is necessary and sufficient to sustain visual transduction, as indicated by ON and OFF transients as signatures of synaptic activity and by visual behavior. Together, these findings support a role of CarT as the transmembrane transporter responsible for the uptake of carcinine into photoreceptor neurons, a critical step in the histamine–carcinine cycle.

Electroretinogram recordings

ERGs were recorded as previously described (Williamson et al., 2010). In brief, voltage measurements of immobilized female flies were recorded with electrodes containing 2M NaCl placed on the corneal surface and inserted into the thorax. Measurements were filtered through an electrometer (IE-210; Warner Instruments, Hamden, CT), digitized with a Digidata 1440A and MiniDigi 1B system, and recorded using Clampex 10.2 and quantified with Clampfit software (Molecular Devices, Sunnyvale, CA). Light pulses (1 s) were computer controlled (MC1500; Schott, Mainz, Germany).

Phototaxis assays

For light-choice assays (Benzer, 1967), male flies were collected within 12 hr of eclosion and aged for 3–4 d. Flies were anesthetized briefly with CO2, transferred to empty culture tubes, and left to recover for 1 hr. For testing, a group of 20 flies was introduced into a T-maze apparatus and allowed to distribute for 20 s between a dark tube and a tube exposed to incandescent light. Each group was tested in triplicate. A light preference index was calculated using the equation, PI = (#Light- #Dark) / #total. For all genotypes, at least 100 flies were tested.

Carcinine uptake assay

Because radioactive carcinine is not commercially available, we developed an immunofluorescence-based transport assay. Drosophila S2 cells were cultured using standard methods and transfected with plasmids containing Myc-tagged constructs as noted using the TransIT-2020 manufacturer’s protocol (Mirus, Madison, WI). Transfected cells were incubated with 0.5 mM carcinine for 24 hr prior to a 1 hr fixation in ice cold 4% 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (wt/vol, Sigma) in 0.1 M phosphate buffer solution. Cells were then permeabilized in phosphate-buffered saline containing 0.3% Saponin, blocked in 5% normal goat serum, and incubated overnight with carcinine and Myc antibodies. Quantification was performed in ImageJ by normalizing the integrated density of the carcinine signal by that of the Myc signal.

Phylogenetics

CarT was identified as a SLC22 family member using BLASTpsi alignments against the NCBI non-redundant protein sequence database. Drosophila SLC22-related family members were identified from existing annotations on FlyBase and by running a BLASTp search with the CarT Isoform C peptide sequence against only Drosophila melanogaster (taxid:7227). Searches for individual organisms were performed using the following taxa identifiers on NCBI BLASTp suite: Homo sapiens (taxid:9606), Mus musculus (taxid:10090), Apis mellifera (taxid:7460). Additionally, previously identified mammalian SLC22 transporters were included (Koepsell, 2013; Zhu et al., 2015; Martin and Krantz, 2014). For preparation of multiple alignments, CLUSTAL Omega was used (http://www.ebi.ac.uk/Tools/msa/clustalo/) with default parameters (Sievers et al., 2011). Multiple sequence alignments were entered into Clustal W2 and phylogenies were generated using the Neighbor-joining clustering method with default parameters (Larkin et al., 2007). The resulting phylogenies were analyzed using Interactive Tree of Life (iToL; http://itol.embl.de), an online phylogeny tree viewer and editor (Letunic and Bork, 2011).

Statistics

Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s or Bonferroni’s multiple comparisons using GraphPad Prism 6.

Decision letter

Hugo J Bellen

Reviewing Editor; Howard Hughes Medical Institute, Baylor College of Medicine, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for submitting your work entitled "The carcinine transporter CarT is required in Drosophila photoreceptor neurons to sustain histamine recycling" for peer review at eLife. Your submission has been favorably evaluated by K VijayRaghavan (Senior editor), Hugo Bellen (Reviewing editor), and two reviewers, one of whom David Krantz, has agreed to reveal his identity.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

Summary:

The authors first performed an RNAi screen to knock down 132 transporter candidates and found that one – CG9317 – which they rename carT, disrupts ERGs a standard measure of signaling in the visual system. They determined that expression was required in photoreceptors but not glia, then confirm their findings using CRISPR/CAS-9 mutants in carT and genetic rescue with a cart transgenes. They back up their electrophysiological results with both behavioral assays demonstrating phototactic deficits and carcinine build-up in carT mutants, both rescued with a carT transgene. Finally they prove that carT transports carcinine into S2 cells as predicted. The paper by Stenesen et al. very convincingly shows that the CarT transporter is required for carcinine uptake, which in turn is a reaction product of histamine modification in glia cells that take up histamine from the synaptic cleft. With this discovery, the circle for release and reuptake of this neurotransmitter has been closed for the first time.

Minor points that we would like to see addressed:

Minor concerns include some difficulty in visualizing the label for carcinine and histamine in Figure 3. It seems likely that this could result from the relatively low resolution of the PDF. Nonetheless, the authors should ensure that the images will be easily interpreted in the final version on the manuscript.

The authors should also comment on the overall distribution of endogenous carT, and whether or not this is known. The model in Figure 4 indicates that carT localizes to photoreceptors but not glia and this is clearly supported by their RNAi data. However, it seems possible that carT could be expressed in other neurons in the CNs and perhaps play a role in other organs.

Additional, small changes that might improve the paper for the interested reader include a list of the genes that were probed in their RNAi screen, and a brief note about the phylogeny of SLC22A family in the fly. The authors state: "[…] our data argue against such redundancy for CarT as it efficiently blocks the histamine-carcinine cycle." Nonetheless, it would be nice to know if there are any similar fly orthologs with the potential to compensate for loss of carT.

I think it would be reasonable to ask the authors to provide better colocalization data for CarT in the synaptic region of the lamina to support their core argument. Ideally one would like to know whether CarT resides at the capitate projections, invaginations of glia into the photoreceptor terminals, but an immuno-EM study of this is beyond the scope of the present work. However, colocalization analysis with glia markers, active zone markers and pre- and postsynaptic cell markers at high resolution would be helpful.

Similarly, it would be helpful to see carcinine accumulations in the mutant indeed occurring the glia cells, as predicted by the model, to support the core argument. Again, this would only require higher resolution and more detailed immunohistochemical analysis.

Author response

Minor points that we like to see addressed:

Minor concerns include some difficulty in visualizing the label for carcinine and histamine in Figure 3. It seems likely that this could result from the relatively low resolution of the PDF. Nonetheless, the authors should ensure that the images will be easily interpreted in the final version on the manuscript.

Indeed, the PDFs for the initial submission were too compressed; looking at the current images we are confident that the data presented in the final version will be easily interpreted. To aid the readers in interpreting the panels addressing carcinine accumulation in carT null flies (Figure 3C,D in revised version), we have included inserts with enlarged regions of similar positions from the lamina of the OreR, carT43, and carT43;longGMR>MycCarT genotypes. This will allow for a more focused comparison of this region, which displays increased carcinine staining in our carT mutant that is rescued by photoreceptor expression of the tagged carT transgene. Also, as outlined in our last comment below, we include an additional panel (Figure 3E in the revised version), which displays higher resolution images of the laminal carcinine increase in carT43 compared to a heterozygous control.

The authors should also comment on the overall distribution of endogenous carT, and whether or not this is known. The model in Figure 4 indicates that carT localizes to photoreceptors but not glia and this is clearly supported by their RNAi data. However, it seems possible that carT could be expressed in other neurons in the CNs and perhaps play a role in other organs.

We now point out in the Results section that in adult flies carT RNA expression is highly enriched in the eye (subsection “CarT is enriched close to photoreceptor synapses”). Furthermore, to more accurately assess CarT distribution, we have now generated a tdTomato-CarT fusion in the endogenous genomic locus using CRISPR/Cas-9 mediated excision coupled with homology directed repair. In the current manuscript, we use ERG recordings to demonstrate that this allele is functional when heterozygous over the carT43 null allele (Figure 2C and D). Localization of this tdTomato-CarT fusion within the visual system is now displayed in Figures 3F–H. In the course of these experiments we gained some initial indications that this transporter is also expressed in other tissues. We feel, however, that a careful description of the cell types expressing CarT in those tissues is beyond the scope of this “Short Report”. We will describe those expression patterns in a separate future manuscript.

Additional, small changes that might improve the paper for the interested reader include a list of the genes that were probed in their RNAi screen, and

The revised manuscript now includes a supplemental table to Figure 1, which lists the genes probed in the RNAi screen, their transporter family, and the stock designation of the specific RNAi line assayed.

a brief note about the phylogeny of SLC22A family in the fly. The authors state: "[…] our data argue against such redundancy for CarT as it efficiently blocks the histamine-carcinine cycle." Nonetheless, it would be nice to know if there are any similar fly orthologs with the potential to compensate for loss of carT.

We now include Figure 2—figure supplement 1, which depicts a phylogenetic comparison of SCL22A family members from the human, mouse, honeybee and Drosophila genomes. This analysis suggests the existence of a distinct invertebrate subclass of SLC22A family members with potential functions similar to CarT. However, as mentioned above, our CRISPR-induced mutants and the transgenic rescue experiments demonstrate carT is the causal gene affecting the identified phenotypes.

I think it would be reasonable to ask the authors to provide better colocalization data for CarT in the synaptic region of the lamina to support their core argument. Ideally one would like to know whether CarT resides at the capitate projections, invaginations of glia into the photoreceptor terminals, but an immuno-EM study of this is beyond the scope of the present work. However, colocalization analysis with glia markers, active zone markers and pre- and postsynaptic cell markers at high resolution would be helpful.

We agree with the reviewers and for that purpose have generated the endogenous tdTomato-tagged carT allele (Figure 3F–H), which we used to perform co-labeling experiments with antibodies against the glial specific Ebony, the synaptic marker, Bruchpilot, and a photoreceptor specific 3xPax3-eGFP transgene (Figure 3G and H). These data further support our argument that the major contribution of CarT to histamine recycling happens locally, close to photoreceptor synapses.

Similarly, it would be helpful to see carcinine accumulations in the mutant indeed occurring the glia cells, as predicted by the model, to support the core argument. Again, this would only require higher resolution and more detailed immunohistochemical analysis.

To address this issue, we performed transgenic labeling of glial cells by expressing a membrane bound uas-mCD8::RFP under Repo-Gal4 control in the carT43 background and localized carcinine by immunohistochemistry (Figure 3D). These data show that carcinine accumulates in and around glia consistent with a buildup of carcinine in response to the carT mutant-induced failure to transport carcinine into photoreceptors.

Competing interests

Funding

National Eye Institute (EY010199, EY021922)

National Institute on Drug Abuse (DA007290)

National Science Foundation (1000176311)

Andrew T Moehlman

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We would like to thank Drs. Hong-Sheng Li and Tao Wang for communicating results before publication, and Drs. Aylin Rodan, Anna Ziegler and Bernhard Hovemann and the Krämer lab for helpful comments and the Bloomington Stock Center for fly stocks, the Berkeley Drosophila Genome Project for producing DNA clones, the Drosophila Genomics Resource Center for their distribution. FlyBase provided important information used in this work. This work was supported by grants from the NIH (EY010199 and EY021922) to H. K., NIH training grant (5T32DA007290) to D.S., NSF Graduate Research Fellowship (1000176311) to A.T.M., and by core grant EY020799.

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